Radioiodinated phospholipid ether analogs and methods of using the same

ABSTRACT

The present invention provides improved radioiodinated phospholipid ether analogs which demonstrate significant tumor avidity and longer plasma half-life than shorter-chain analogs. The radioiodinated phospholipid ether analogs of the present invention provide superior imaging and visualization of neoplastic lesions and tumor-specific cytotoxic cancer therapy.

This is a Divisional of application Ser. No. 09/319,406, filed Jun. 4,1999, now U.S. Pat. No. 6,255,519 which claims benefit under 35 U.S.C.§119 to PCT/US96/19352, filed on Apr. 12, 1999, both of which are hereinincorporated by reference in their entirety for all purposes.

This invention was made with government support under grant CA08349 fromthe National Institutes of Health. The United States Government hascertain rights in the invention.

FIELD OF THE INVENTION

The present invention relates generally to the field ofradiopharmaceuticals and biological probes, and more specifically toradiolabelled analogs of phospholipid ethers useful in cancer diagnosisand treatment.

BACKGROUND OF THE INVENTION

The early detection of cancer has been one of the primary goals ofmodern imaging technology, since the identification of a suspected tumorin a localized stage significantly improves the chances for successfultreatment and elimination of the cancerous tissue. A large number ofimaging strategies have therefore been designed, using a variety oftechniques and modalities, to aid the physician in making an accuratediagnosis as early as possible.

Unfortunately, conventional imaging techniques such as computerizedtomography (CT) and MRI (magnetic resonance imaging) are limited m theirability to afford a conclusive diagnosis of a suspected lesion, sincethey are only capable of observing differences in the density ormorphology of tissues. A more invasive and costly biopsy procedure isoften necessary to provide a definitive diagnosis. In contrast, nuclearmedicine techniques such as positron emission tomography (PET) andsingle photon emission tomography (SPECT) can provide functional orbiochemical information about a particular organ or area of interest.However, the success of these nuclear imaging techniques depends inlarge part on the selective uptake and detection of appropriateradiopharmaceuticals. Selective uptake, in turn, depends upon thedevelopment of radiopharmaceuticals with a high degree of specificityfor the target tissue. Unfortunately, the tumor-localizing agentsdeveloped thus far for oncological applications have had only limitedapplication.

For example, one of these prior art compounds, ⁶⁷Ga gallium citrate, wasoriginally identified for its ability to accumulate in tumor tissue.Unfortunately, ⁶⁷Ga gallium citrate is taken up by a variety of othernon-tumorous lesions as well, including inflammatory lesions, andunacceptable amounts of radioactivity can also accumulate in liver andspleen tissue. The rapid buildup of a radiopharmaceutical in theseorgans can seriously interfere with the imaging of nearby lesions andalso negatively impacts the dosage that can safely be given to apatient.

An alternative approach has been to develop radiolabelled monoclonalantibodies (Mabs) directed to tumor-specific antigens. However, thesemonoclonal antibodies are specific only to the particular tumor tissuefor which they have been produced, and therefore will not localizegenerally in neoplastic tissue. Moreover, the use of Mabs for diagnosticimaging has lead to additional problems, including varying degrees ofantigen expression, low tumor uptake, non-specific binding and adverseimmunogenic reactions.

In an attempt to address these problems, the present inventors haverecently identified and developed a series of novel compoundsdemonstrating useful tumor specificity. See, e.g., U.S. Pat. Nos.4,925,649; 4,965,391; 5,087,721; and 5,347,030; all of which are hereinincorporated by reference. It is believed that these radioiodinatedphospholipid ether analogs take advantage of a unique biochemicalcharacteristic of malignant tumor cells; i.e. the large concentration ofnaturally-occurring ether lipids in the cell membranes relative tocorresponding normal tissues. Although the precise mechanism of actionis not fully understood, the prevailing hypothesis is that thephospholipid ether analogs become entrapped in tumor membranes.Accordingly, these compounds localize in tumor tissue and remain inplace for diagnostic and/or therapeutic applications.

The selective retention of the radiolabelled phospholipid ether analogsdescribed in the above patents has been demonstrated in a variety ofrodent and animal tumors. Unfortunately, the data obtained from thesestudies has also demonstrated a relatively rapid clearance of theradiopharmaceutical compound from the blood, and an undesirableaccumulation by non-target tissues. As noted above, non-target tissueuptake can decrease the efficacy of radiodiagnostic imaging by creatinghigh background activity, or by causing excessive exposure ofradiosensitive tissues to the injected radioactivity.

Accordingly, there remains a significant need in the art forradiopharmaceuticals which exhibit a rapid clearance from non-targettissues as well as an extended half-life in the blood plasma, whilestill retaining its specificity and avidity for neoplastic tissue. Suchan agent should not only assist in the non-invasive imaging of primarytumors and metastases, but should also provide a potential cytotoxicagent for site-specific eradication of the tumor tissue.

SUMMARY OF THE INVENTION

The present invention solves the problems present in the prior artthrough the provision of improved radiolabelled phospholipid etheranalogs of naturally-occurring phospholipid ether compounds, having thegeneral Formula I:

where X is a radioactive isotope and n is an integer between 16 and 30.In a preferred embodiment of the present invention, X is a radioactiveisotope of iodine, preferably selected from the group comprising ¹²³I,¹²⁵I, and ¹³¹I. It is further contemplated that X can be substituted atthe ortho, meta or para position on the aromatic ring. In a preferredembodiment, the radioactive isotope is substituted at the para position.

Y is selected from the group comprising H, OH, COOH,

and OR, and Z is selected from the group comprising NH₂, NR₂, and NR₃,wherein R is an alkyl or aralkyl substituent.

In accordance with a specific illustrative embodiment of the invention,the improved compound is1-O-[18-(p-iodophenyl)octadecyl]-1,3-propanediol-3-phosphocholine.

In accordance with another specific illustrative embodiment, theimproved compound is1-O-[18-(p-Iodophenyl)octadecyl]-2-O-methyl-rac-glycero-3-phosphocholine:

In a further embodiment of the invention, the radiolabelled aralkyl sidechain may be substituted directly onto the alkyl phosphocholine moietyin accordance with general Formula II:

where X is a radioactive isotope substituted at the ortho, meta or paraposition, preferably of iodine, n is an integer from 16 to 30, and Y isselected from the group comprising NH₂, NR₂ and NR₃, wherein R is analkyl or aralkyl substituent.

In accordance with a specific illustrative embodiment of the invention,the improved compound is 18-(p-iodophenyl)octadecyl phosphocholine.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is an illustrative preparatory scheme for18-(p-iodophenyl)octadecanol;

FIG. 2 is an illustrative preparatory scheme for18-(p-iodophenyl)octadecyl phosphocholine;

FIG. 3 is an illustrative preparatory scheme for1-O-[18-(p-iodophenyl)octadecyl]-1,3-propanediol-3-phosphocholine;

FIG. 4 is an illustrative preparatory scheme for1-O-[18-(p-iodophenyl)octadecyl]-2-O-methyl-rac-glycero-3-phosphocholine;

FIGS. 5A-5C provide the chemical structures of the alkyl chain lengthanalogs 12-(p-iodophenyl)dodecyl phosphocholine (FIG. 5A),15-(p-iodophenyl)pentadecyl phosphocholine (FIG. 5B), and18-(p-iodophenyl)octadecyl phosphocholine (FIG. 5C) used inbiodistribution studies with rats bearing Walker-256 tumor;

FIG. 6 provides a line graph illustrating the blood clearance profile of12-(p-iodophenyl)dodecyl phosphocholine (C-12),15-(p-iodophenyl)pentadecyl phosphocholine (C-15) and18-(p-iodophenyl)dodecylphosphocholine (C-18) in rats bearing Walker-256tumors;

FIGS. 7A-7C provide the chemical structures of the alkyl chain lengthanalogs 12-(p-iodophenyl)dodecyl phosphocholine (FIG. 7A),18-(p-iodophenyl)octadecyl phosphocholine (FIG. 7B) and1-O-[18-(p-iodophenyl)octadecyl]-1,3-propanediol-3-phosphocholine usedin biodistribution studies with rats bearing Dunning (MATLyLu) prostatetumors;

FIGS. 8A-8C are depictions of whole-body gamma-camera scintigraphy scansof Dunning (MATLyLu) prostate tumor-bearing using18-(p-iodophenyl)octadecyl phosphocholine (FIG. 8A),12-(p-iodophenyl)dodecyl phosphocholine (FIG. 8B) and1-O-[18-(p-iodophenyl)octadecyl]-1,3-propanediol-3-phosphocholine (FIG.8C).

GENERAL DESCRIPTION OF THE INVENTION

The present invention represents a significant improvement of theradiolabelled phospholipid ether analogs previously described in theprior art, providing a series of radiopharmaceutical compoundsexhibiting greatly increased plasma half-life and a significantly loweraccumulation in non-target organs. Thus, these improvedradiopharmaceuticals provide superior tumor imaging capabilities byreducing the amount of background radiation from non-target tissues, andthe rapid clearance of the compounds from non-target organs also allowsfor an increase in the radiation dosimetry of the radiopharmaceutical,for further enhancement of tumor imaging capabilities and cytotoxiccancer therapy.

Surprisingly, the nature of the phospholipid ether compounds whichexhibit these enhanced capabilities are compounds having an extension ofthe carbon chain bearing the radiolabelled phenyl group. Previousstudies with related alkyl phosphocholine analogs had demonstrated thatblood levels actually decreased with increasing chain length, whiletumor levels increased. See, e.g., Kotting et al.,“Alkylphosphocholines: influence of structural variation onbiodistribution at antineoplastically active concentrations.” CancerChemother. Pharm. 30:105-112 (1992). In contrast, the improved compoundsof the present invention have displayed a propensity to remain in thecirculation much longer than the original shorter chain analogs, and thedelayed clearance from the blood plasma advantageously results inadditional opportunities for uptake of the radiopharmaceuticals by tumortissue as they continuously circulate through the vasculature.

Although an understanding of the underlying mechanism is not essentialto the beneficial use of the improved compounds provided by the presentinvention, the inventors believe that the uptake and transport of theradiolabelled analogs by plasma components may be an important factorrelating to the tumor retention of the compounds. Certainly, increasingthe length of the carbon chain results in a corresponding increase inthe lipophilicity of the analogs, and greater lipophilicity wouldpresumably increase the affinity of these compounds for the cellmembrane. There is also evidence (not shown) to indicate that the longercarbon chain alters the binding affinity of these compounds for plasmacomponents such as plasma albumin.

The differences in the clearance and quantity of radioactivity innon-target tissues observed with the improved compounds of the presentinvention significantly enhances the chances for the imaging of tumorsin human patients. Moreover, it should also be noted that the improvedphospholipid ether analogs of the present invention are cytotoxic totumor cells, even without the presence of a radioactive isotope in thecompound. Therefore, the inclusion of a long-lived radioactive isotopeof iodine, for example, yields tumor-specific radiopharmaceuticals whichare tissue-destructive by more than one mode.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following examples relate to specific embodiments and methods ofusing the improved radiolabelled phospholipid analogs of the presentinvention, and include illustrative methods for synthesizing theanalogs. All starting materials and reagents were obtained from AldrichChemical Company, Milwaukee, Wis.

In the experimental disclosure which follows, the followingabbreviations apply: eq (equivalents); M (Molar); μM (micromolar); N(Normal); mol (moles), mmol (millimoles); μmol (micromoles): nmol(nanomoles); g (grams); mg (milligrams); kg (kilograms); μg(micrograms); L (liters); ml (milliliters); μl (microliters); cm(centimeters); mm (millimeters); μm (micrometers); nm (nanometers); ° C.(degrees Centigrade); h (hours): min (minutes); sec (seconds): msec(milliseconds); Ci (Curies) mCi (milliCuries); μCi (microCuries); TLC(thin layer achromatography); Ts (tosyl); Bn (benzyl); Ph (phenyl); Ms(mesyl); Et (ethyl), Me (methyl).

EXAMPLE 1

The synthesis of 18-(p-Iodophenyl)octadecanol (Compound VI), which is apreliminary compound for the illustrative preparatory schemes for thephospholipid ether analogs discussed in detail in Examples 2 through 4below, was accomplished from commercially-available15-(p-iodophenyl)pentadecanoic acid (Compound I) in accordance with theillustrative preparatory scheme shown in FIG. 1.

In general terms, aliphatic chain elongation from C-15 to C-18 wasachieved by Li₂CuCl₄-catalyzed cross-coupling between Grignard reagent(Compound IV) prepared from benzyl 3-bromopropyl ether and15-(p-iodophenyl)pentadecyl tosylate (Compound III). See, e.g., Fouquetand Schlosser. “Improved carbon-carbon linking by controlled coppercatalysis,” Angew. Chem. Int. Ed. 13:82-83 (1974); Schlosser andBossert, “The ‘two-fold reaction’ benchmark applied to the coppercatalyzed assembling of 1,ω-difunctional hydrocarbon chains,”Tetrahedron 47:6287-92 (1991). Cleavage of the benzyl protective groupin Compound V by anhydrous aluminum chloride-anisole (see, e.g., Akiyamaet al., “AlCl₃-N,N-dimethylaniline: a novel benzyl and allyl ethercleavage reagent.” Bull. Chem. Soc. Jpn. 65:1932-38 (1992)) affordedalcohol VI with 18 carbon atoms in aliphatic chain.

Specifically, to a solution of 15-(piodophenyl)dodecanoic acid (CompoundI) (210 mg; 0.47 mmol) in 3 ml of tetrahydrofuran was added BH₃.THFcomplex (1 ml of 1.0 M solution; 1 mmol) dropwise at 0° C. The reactionmixture was then stirred at room temperature under anhydrous conditionsfor 10 h. It was again cooled to 0° C. and quenched with water. Ethylacetate and additional water were added. The organic layer wasseparated, washed with water and dried (Na₂SO₄). Evaporation of solventgave the white solid Compound II, 15-(p-iodophenyl)pentadecanol (204 mg,100%). Compound II was used in the next step without purification.

A mixture of 15-(p-iodophenyl)pentadecanol (Compound II) (200 mg; 0.47mmol), tosyl chloride (102 mg; 0.53 mmol) and 4-dimethylaminopyridine(66 mg; 0.53 mmol) in dichloromethane (3 ml) was stirred at roomtemperature for 12 h. The reaction mixture was then partitioned betweenchloroform (40 ml), methanol (40 ml) and 0.1 N hydrochloric acid (36ml). The chloroform layer was separated and extraction was repeated(2×40 ml of chloroform). Extracts were combined, dried (Na₂SO₄) andevaporated. The residue was chromatographed on silica gel inhexane-chloroform (from 95:5 to 40:60) to give Compound III,15-(p-Iodophenyl)pentadecyl tosylate (259 mg; 95%).

To a solution of 3-benzyloxypropanol (5.28 g; 31.8 mmol) and carbontetrabromide (3.1 g; 39.6 mmol) in dichloromethane (50 ml) was slowlyadded triphenylphosphine (10.4 g; 39.7 mmol) at 0° C. The reactionmixture was stirred at 0° C. for 30 min and at room temperature for 1 h.By that time TLC in chloroform showed completion of the reaction. Mostof the solvent was evaporated, the residue was diluted with hexane (200ml) and the precipitate of triphenylphosphinoxide was removed byfiltration. Evaporation of the filtrate gave an oily residue which waspurified by chromatography in 1% ether in hexane. This afforded CompoundIV, benzyl 3-bromopropyl ether (5.61 g; 77%).

To a suspension of magnesium powder (60 mg: 2.5 mmol) in tetrahydrofuran(1.5 ml) was added dibromoethane (0.02 ml) for activation. After thereaction with dibromoethane had ceased, the solution was removed bysyringe and replaced with fresh tetrahydrofuran (2.5 ml). Then, benzyl3-bromopropyl ether (Compound IV) (0.12 ml; 0.67 mmol) was addeddropwise to the stirred suspension of magnesium for 30 min. When all thehalide had been added, stirring was continued for an additional 2 h atroom temperature. The green-gray solution of Grignard reagent wascarefully withdrawn by syringe and transferred to 25 ml flask, which wasthen cooled in a dry ice bath to −78° C. A solution of Li₂CuCl₄ (0.0067mmol/ml) in tetrahydrofuran (0.5 ml; 0.0034 mmol) was added to theGrignard reagent under stirring, followed by Compound III,15-(p-iodophenyl)pentadecyl tosylate (260 mg; 0.44 mmol) in 3 ml oftetrahydrofuran.

The reaction mixture was allowed to warm to room temperature during a 2h period, and stirring was continued for an additional 12 h. Thereaction was quenched by ammonium chloride solution and extracted withethyl acetate. The extract was washed with water, dried (Na₂SO₄) andevaporated. Silica gel chromatography with 2% ether in hexane providedCompound V, 1-Benzyloxy-18-(p-iodophenyl)octadecane (190 mg; 76%).

Finally, to a solution of 1-benzyloxy-18-(p-iodophenyl)octadecane(Compound V) (185 mg; 0.33 mmol) and anisole (0.15 ml; 1.32 mmol) indichloromethane (3 ml) was added anhydrous aluminum chloride (135 mg; 1mmol) at the room temperature. Stirring was continued for 2 h. Thereaction was quenched by diluted hydrochloric acid and extracted bychloroform. Extract was dried (Na₂SO₄) and evaporated. The residue waschromatographed in hexane-ethyl acetate (gradient from 95:5 to 85:15) togive 18-(p-iodophenyl)octadecanol, Compound VI (123 mg; 77%).

EXAMPLE 2

Further conversion of 18-(p-iodophenyl)octadecanol into the desiredphosphocholines was performed as described in detail for the C-12analogs in Rampy et al., “Synthesis and biological evaluation ofradio-iodinated phospholipid ether analogs,” Nucl. Med. Biol. 22,505-512 (1995). In one preferred embodiment, the improved phospholipidether analog contemplated by the present invention is a simple straightchain alkyl phospholipid, 18-(p-iodophenyl)octadecyl phosphocholine(Compound XVI). The synthesis of Compound XVI was accomplished accordingto the illustrative preparatory scheme shown in FIG. 2.

As illustrated in FIG. 2, 2-chloro-2-oxo-1,3,2-dioxaphospholane (0.025ml; 0.27 mmol) was added to the stirred solution of18-(p-iodophenyl)octadecanol (Compound VI) (115 mg; 0.24 mmol) in drybenzene (3 ml) containing triethylamine (0.042 ml; 0.29 mmol). Stirringwas continued overnight. The precipitated triethylamine hydrochloridewas filtered off and the solvent was removed in vacuo. The residue wastransferred into a pressure bottle. A solution of trimethylamine inacetonitrile (5 ml; 25% w/v) was added. The bottle was sealed and heatedat 75° C. for 24 h. The acetonitrile was then evaporated and the residuewas chromatographed on silica gel with chloroform-methanol (gradientfrom 10:0 to 5:5), followed by final elution withchloroform-methanol-water (65:25:4). After evaporation of the solvent,the product was precipitated by addition of acetone to give a whitesolid (130 mg; 84%).

EXAMPLE 3

In another preferred embodiment, the improved phospholipid ether analogscontemplated by the present invention are constructed using apropanediol backbone. In this example, the synthesis of1-O-[18-(p-Iodophenyl)octadecyl]-1,3-propanediol-3-phosphocholine(Compound XIV) was accomplished according to the illustrativepreparatory scheme shown in FIG. 3.

To a solution of 18-(p-iodophenyl)octadecanol (Compound VI) (150 mg;0.317 mmol) and triethylamine (0.07 ml; 0.475 mmol) in dry methylenechloride (2 ml) was added methane sulfonyl chloride (0.03 ml: 0.38 mmol)at 0° C. Stirring was continued for 40 min and the reaction was quenchedby addition of water. The reaction mixture was diluted with chloroformand washed several times with water. The chloroform layer was dried(Na₂SO₄) and evaporated. The residue was chromatographed in hexane-ethylacetate (9:1). This afforded pure Compound VII,18-(p-Iodophenyl)octadecyl methanesulfonate (142 mg; 82% yield).

To a solution of 3-benzyloxy propanol (Compound VIII) (0.03 ml; 0.18mmol) and 18-(p-iodophenyl)octadecyl methanesulfonate (Compound VII) (66mg: 0.12 mmol) in dry dimethylformamide (3 ml) was added sodium hydride(8 mg of 60% suspension in oil; 0.2 mmol) at the room temperature. Thereaction mixture was stirred for 12 hr, quenched by water and extractedwith ethyl acetate. The extract was washed with brine, dried andevaporated. Column chromatography in hexane-ethyl acetate (gradient from95:5 to 85:15) afforded Compound X,1-O-[18-(p-Iodophenyl)octadecyl]-3-O-benzyl-1,3-propanediol (60 mg; 81%yield).

Compound X was debenzylated by AlCl₃-anisole as described for thesynthesis of Compound VI, to produce Compound XII,1-O-[18-(p-Iodophenyl)octadecyl]-1,3-propanediol (50 mg; 0.08 mmol), (42mg, 99% yield). Finally, the desired phosphocholine XIV was synthesizedabove from the alcohol XII (42 mg; 79 mmol) in an analogous manner tothat described in Example 2 for Compound XVI, (45 mg, 55% yield).

EXAMPLE 4

This example illustrates yet another preferred embodiment of theimproved phospholipid ether analogs contemplated by the presentinvention, wherein the hydrogen located at the 2-position of CompoundXIV is replaced with an O-methyl group. The synthesis of1-O-[18-(p-Iodophenyl)octadecyl]-2-O-methyl-rac-glycero-3-phosphocholine(Compound XV) was accomplished according to the illustrative preparatoryscheme shown in FIG. 4.

CompoundXI,1-O-[18-(p-Iodophenyl)octadecyl]-2-O-methyl-3-O-benzyl-rac-glycerol,was synthesized as described above in Example 3 above for Compound X,from 18-(p-iodophenyl)octadecyl methanesulfonate (Compound VII) (67 mg)and 1-O-benzyl-2-O-methyl-rac-glycerol (Compound IX) (36 mg), (62 mg,78% yield). The synthesis of Compound IX is known in the art and isdescribed in detail in Pinchuk et al., Chem. Phys. Lipids, 59:263-65(1991). Briefly, to 2-O-methyl-1,2-O,O-benzylideneglycerol (2.4 g, 12.4mmol) cooled in an ice bath was added 18 ml of 1.0 M borane-THF solution(18 ml, 18 mmol) with stirring. After 10 minutes the mixture was left atroom temperature for 12 h, then for 48 h at 40-45° C. until TLC(hexane/ethyl acetate 6:4) showed no starting material remained. Themixture was cooled and water was added drop-wise to destroy the boraneexcess. The solvent was evaporated, the residue was dissolved in water,extracted with ether (3×100 ml), extracts were washed with water, dried(Na₂SO₄), and evaporated to give 2.32 g (96%) of a slightly yellow oil.The compound was used for the following step without any furtherpurification.

Compound XIII, 1-O-[18-(p-Iodophenyl)octadecyl]-2-O-methyl-rac-glycerol,was synthesized from the benzyl ether (Compound XI) (58 mg, 0.09 mmol)by the procedure described for Compound VI in Example 1. (40 mg, 80%yield). The desired phosphocholine XV was synthesized from the alcoholXIII (33 mg, 0.06 mmol) by the procedure described in Example 3 abovefor Compound XVI, (32 mg 75% yield).

EXAMPLE 5 Radioiodination of Phospholipid Ether Analogs

For certain uses, such as scintigraphy or experimental evaluation oftissue distribution, it is desirable to create radioactive compounds.Radioiodination of the iodinated phospholipid ether analogues disclosedherein, or one of the intermediates in the synthesis pathway, can beaccomplished by a variety of techniques, some of which are known in theart. For example, aromatic compounds with electron donating groups (suchas anilines) can be radiolabelled by electrophilic iodination in thepresence of radioiodine, iodine monochloride, chloramine-T, iodogen,etc. Unactivated aromatic rings can be radioiodinated by exchange of aleaving group, such as aryl boronic acids, aryl thalliumtrifluoroacetates, triazenes or metallated arenes with radioiodine.Direct electrophilic radioiodination of a phenyl ring is yet anotheralternative, but may produce isomeric mixtures which are difficult toseparate. Iodine exhange of aryl iodides with radioiodine may be apreferable approach insofar as no complex separation techniques arenecessary, since the substrate and radioiodinated product are chemicallyidentical.

In a preferred embodiment of the invention, an isotope exchange-typetechnique is utilized wherein the substrate and radioiodine are reactedat an elevated temperature in a “melt.” The molten reaction mediumpossesses a sufficiently high dielectric constant to solubilize both thesubstrate and radioiodide. Examples of reaction media currently in useare benzoic acid (mp 122° C., bp 249° C.) and acetamide (mp 182° C., bp221° C.). In a specific preferred embodiment, an acidic exchange mediumcomprising pivalic acid, a homolog of acetic acid, also known astrimethyl acetic acid, can be used. Pivalic acid has a melting point of33° C. and a boiling point of 164° C.

The phospholipid ether analogs discussed herein were made via isotopeexchange in pivalic acid. This technique is described in detail inWeichert et al., “Radioiodination via isotope exchange in pivalic acid,”Appl. Radiat. Isot. 37:907-13 (1986). Briefly, the unlabeled compound(0.5 mg) was placed in a 300-μl V-vial (Wheaton, Millville, N.J.) fittedwith teflon faced seal and screw cap. Absolute ethanol (20 μl) was addedvia a microliter syringe, followed by aqueous Na¹²⁵I (0.5-3.0 mCi, 2-10μl) (no-carrier-added in reductant-free 0.1 N NaOH from AmershamRadiochemicals). The vial was gently swirled to dissolve the contentsand ensure homogeneity.

Inlet and outlet cannuli were inserted and a gentle stream of nitrogenwas applied to remove the solvents. Two successive in-line charcoaltraps were placed on the outlet side in order to trap any volatileradioiodine present in the reaction vial. Once dry, solid pivalic acid(5 mg), previously dried by azeotropic removal of water with toluene anddistilled under nitrogen was added. The vial was sealed and heated at160° C. in a preheated single well aluminum heating block containing 1cm of sand in the bottom of the well. After 1 hr., the reaction vial wasremoved from the heating block and allowed to cool to room temperature.Absolute ethanol (70 μl) was added through a micro syringe followed bygentle agitation and subsequent removal of a TLC sample (1-2 μl).

The entire contents of the reaction vial were then injected directlyonto a silica gel HPLC column eluted with hexane/isopropanol/water(40:52:8) at 0.8 ml/min. Peaks were analyzed by both UV (230 and 254 mn)and radiodetection. After pooling appropriate fractions theradiochemical purity of the final product was monitored by TLC (gammaand UV detection) and by HPLC (UV at 230254 nm and radiochemicaldetector). Fractions were combined and the solvent was removed with agentle stream of nitrogen. HPLC analysis of the final compound confirmedboth chemical (UV at 230/254 nm) and radiochemical (radioactivity)purity.

Of course, any isotope of iodine such as the clinically used isotopes,¹²²I, ¹²³I, ¹²⁵I and ¹³¹I can be used. ¹²⁵I is preferred for in vitrowork in the laboratory due to its relatively long half-life. Forradiodiagnostic purposes in humans, ¹²³I or ¹³¹I are preferred due totheir shorter half-lives and favorable imaging energies. Thus, theradioiodination procedure described above may be modified, as known bythose of skill in the art, to compensate for the difference inhalf-life.

It is further contemplated that the radioiodinated phospholipid etheranalogs of the present invention may be solubilized in a suitabletransport agent or carrier vehicle, and administered to mammaliansubjects as radiologic agents by any known manner, preferably parentallysuch as intravenously or intraperitonally. It is not intended that thepresent invention be limited by the particular nature of the therapeuticpreparation. For example, such compositions can be provided togetherwith physiologically tolerable liquid, gel or solid carriers, diluents,adjuvants and excipients.

These therapeutic preparations can be administered to mammals forveterinary use, such as with domestic animals, and clinical use inhumans in a manner similar to other therapeutic agents. In general, thedosage required for therapeutic efficacy will vary according to the typeof use and mode of administration, as well as the particularizedrequirements of individual hosts.

Such compositions are typically prepared as liquid solutions orsuspensions, or in solid forms. Oral formulations for cancer usuallywill include such normally employed additives such as binders, fillerscarriers, preservatives, stabilizing agents, emulsifiers, buffers andexcipients as, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharin, cellulose, magnesiumcarbonate, and the like. These compositions take the form of solutions,suspensions, tablets, pills, capsules, sustained release formulations,or powders, and typically contain 1%-95% of active ingredient,preferably 2%-70%.

The compositions are also prepared as injectables, either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid prior to injection may also be prepared. Theradioiodinated compounds of the present invention are often mixed withdiluents or excipients which are physiologically tolerable andcompatible. Suitable diluents and excipients are, for example, water,saline, dextrose glycerol, or the like, and combinations thereof. Inaddition if desired the compositions may contain minor amounts ofauxiliary substances such as wetting or emulsifying agents, stabilizingor pH buffering agents.

Additional formulations which are suitable for other modes ofadministration, such as topical administration, include salves,tinctures, creams, lotions, and, in some cases, suppositories. Forsalves and creams, traditional binders, carriers and excipients mayinclude, for example, polyalkylene glycols or triglycerides.

EXAMPLE 6 Biodistribution Studies with Walker-256 Carcinosarcoma

The three radiolabelled compounds illustrated in FIG. 5, including theimproved C-18 analog 18-(p-iodophenyl)octadecyl phosphocholine (CompoundXVI) described in Example 2, were prepared and administered to two setsof female Sprague Dawley rats (Charles River, Portage, Mich.). Onenormal set was used as a control, and one set was inoculated withWalker-256 carcinosarcoma cells (5×10⁶ cells) in 0.2 ml saline in theright hindlimb. The Walker carcinoma cell line was provided by Dr. JamesVarani of the Department of Pathology at the University of Michigan, andis an accepted cell line representative of effect in humans. See, e.g.Tayck et al., “Influence of the Walker 256 Carcinosarcoma on Muscle,Tumor, and Whole-Body Protein Synthesis and Growth Rate in theCancer-Bearing Rat, Cancer Res. 46:5649-54 (1986).

The animals were used 6-8 days later when the tumor weight averaged 10g. The radiolabelled compounds were dissolved in absolute ethanol(50-500 μl) and Tween-20 (0.1 ml/mg of compound) was added to thesolution. Ethanol was removed by evaporation under a stream of nitrogen.Physiological saline or sterile water was added, to give a 2-3% Tween-20solution which was subsequently mixed by vortex. The solubilizedradiolabelled compounds (5-10 μCi, 0.3 ml) were administeredintravenously via the tail vein to tumor bearing rats, and the animalswere sacrificed by exsanguination while under ether anesthesia at thevarious time points. Blood samples were collected through cardiacpuncture and selected tissues (liver, kidney, etc.) were removed,trimmed, blotted to remove excess blood and weighed. Large organs werethoroughly minced with scissors to obtain random representative tissuesamples.

For the biodistribution experiments with 12-(p-iodophenyl)dodecylphosphocholine, weighed tissue samples were counted with a wellscintillation counter (85% counting efficiency). The concentration ofradioactivity in each tissue was expressed as a percentage ofadministered dose per gram of tissue.

Tissue distribution was assessed at various times followingadministration of the radioiodinated chain length isomers. Anillustration of the data for each analog is set forth in Table 1 below.The results are expressed as the mean % administered dose per gram±SEM(% Dose/g±SEM).

C-12 Analog C-15 Analog C-18 Analog Tissue 24 hr 48 hr 24 hr 48 hr 24 hr48 hr Adrenal 0.33 ± 0.01 0.29 ± 0.01 0.73 ± 0.08 0.49 ± 0.05 0.85 ±0.04 0.92 ± 0.06 Blood 0.27 ± 0.04 0.27 ± 0.03 0.16 ± 0.01 0.14 ± 0.010.96 ± 0.00 0.64 ± 0.07 Duodenum 1.35 ± 0.13 0.77 ± 0.06 1.13 ± 0.131.38 ± 0.24 0.69 ± 0.05 0.68 ± 0.04 Kidney 4.10 ± 0.44 3.44 ± 0.08 1.14± 0.11 0.91 ± 0.04 0.65 ± 0.00 0.59 ± 0.04 Liver 1.62 ± 0.17 1.37 ± 0.011.29 ± 0.10 0.83 ± 0.04 0.55 ± 0.04 0.59 ± 0.05 Lung 1.06 ± 0.04 0.53 ±0.01 0.97 ± 0.03 0.81 ± 0.04 0.88 ± 0.06 0.76 ± 0.07 Plasma 0.39 ± 0.050.39 ± 0.05 0.16 ± 0.01 0.15 ± 0.03 1.47 ± 0.03 0.95 ± 0.11 Tumor 2.99 ±0.34 1.84 ± 0.09 1.47 ± 0.10 1.65 ± 0.23 0.98 ± 0.07 1.14 ± 0.01

As the above data clearly demonstrates, the clearance of the C18 analogfrom non-target tissues was much more rapid than from tumor, and thequantities of radioactivity detected in the liver, kidney and duodenumwere significantly lower following administration of the C18 analog, ascompared to the same organs in the C15 and C12 analog studies. Inaddition, the C18 analog was retained in the circulation to a muchgreater extent than the other chain length isomers surveyed. As Table 1illustrates, the plasma level for the C-18 analog was significantlyhigher than the plasma levels for both the C-12 and the C-15 analog.Moreover, even at 120 hours, blood levels for the C18 analog were0.60±0.01 (% Dose/g±SEM), as compared to levels of 0.07±0.01 and0.22±0.03 for the C15 and C12 analogs, respectively. This effect isillustrated in FIG. 6, which shows the blood clearance profile of allthree analogs up to 120 hours post-injection.

While the rapid decline in radiation levels in non-target tissue wasaccompanied by a much more subtle reduction in the radioactivity presentin the tumor, the C18 analog still retained a beneficial degree of tumorspecificity in this tumor model. In fact, the level of C18 in the tumorincreased significantly over a longer time period, most likely as aresult of the longer plasma half-life.

EXAMPLE 7 Biodistribution Studies with Dunning Prostate Tumor

The three radiolabelled compounds illustrated in FIGS. 7A-7C, includingthe improved C-18 analogs 18-(p-iodophenyl)octadecyl phosphocholine(Compound XVI—FIG. 7B) and1-O-[18-(p-iodophenyl)octadecyl]-1,3-propanediol-3-phosphocholine(Compound XIV—FIG. 7C) described in Examples 2 and 3, respectively, wereprepared and administered to two sets of male Copenhagen rats (HarlanSprague-Dawley, Indianapolis, Ind.). One group of normal animals wasused as a control, and another group was inoculated with Dunningprostate tumor cells (1×10⁶ MAT-LyLu cells) in 0.2 ml saline in theright hindlimb. The MAT-LyLu subline of the Dunning (R-3327)adenocarcinoma prostate tumor was provided by Dr. Ken Pienta of theDepartment of Urology at the University of Michigan, and is an acceptedcell line representative of effect in humans. See, e.g., Pienta et al.,“Inhibition of Spontaneous Metastasis in a Rat Prostrate Cancer Model byOral Administration of Modified Citrus Pectin, J. Natl. Cancer Inst.87:348-53 (1995).

The animals were maintained with free access to food and water until day8-10. The radioiodinated phospholipid ether analog formulated in 2%Tween 20—sterile water (5-10 μCi, 0.3-0.4 ml, described above in Example6) was administered intravenously into the tail vein of thetumor-bearing animals under metofane anesthesia. The animals wereeuthanized at selected time points after injection (n=3 per time point)and samples of blood, plasma and a variety of tissues including tumorand prostate are collected and analyzed for radioiodine content.Biodistribution values are calculated as the mean of the individual %administered dose per gram of tissue and % dose per organ values. Anillustration of the data for each analog is set forth in Table 2 below.These results are also expressed as the mean % administered dose pergram±SEM (% Dose/g±SEM).

C-12 Analog Compound XIV Compound XVI Tissue 24 hr 48 hr 24 hr 48 hr 24hr 48 hr Adrenal 0.29 ± 0.01 0.23 ± 0.01 1.37 ± 0.03 1.04 ± 0.07 0.88 ±0.02 0.76 ± 0.03 Blood 0.23 ± 0.02 0.22 ± 0.01 0.41 ± 0.01 0.29 ± 0.010.88 ± 0.04 0.64 ± 0.05 Kidney 4.45 ± 0.27 3.38 ± 0.43 0.73 ± 0.02 0.65± 0.01 0.59 ± 0.02 0.51 ± 0.03 Liver 1.27 ± 0.02 1.05 ± 0.03 0.57 ± 0.020.36 ± 0.01 0.57 ± 0.02 0.40 ± 0.03 Lung 0.48 ± 0.03 0.33 ± 0.02 0.89 ±0.07 0.78 ± 0.02 0.84 ± 0.01 0.69 ± 0.05 Plasma 0.32 ± 0.03 0.32 ± 0.010.58 ± 0.01 0.42 ± 0.03 1.43 ± 0.06 0.99 ± 0.11 Tumor 0.37 ± 0.03 0.30 ±0.01 0.43 ± 0.05 0.39 ± 0.01 0.75 ± 0.04 0.81 ± 0.04

As with the Walker-256 tumor in Example 6, the C-18 analogs againdemonstrated kidney and liver levels several times lower than the C-12analog in the prostate tumor model. Importantly, however, the longerchain analogs also demonstrated a superior tumor avidity, with tumorlevels of Compound XVI more than twice as high as the shorter chainanalog. Thus, the improved compounds of the present invention may findparticularly advantageous use in the imaging and/or treatment ofprostate tumors.

EXAMPLE 8 Gamma Camera Scintigraphy Studies with Dunning R3327 ProstateTumor

Comparative in vivo scintigaphic studies were also conducted using theC-12 analog 12-(p-iodophenyl)dodecyl phosphocholine and the C-18 analogs18-(p-iodophenyl)octadecyl phosphocholine (Compound XVI) and1-O-[18-(p-iodophenyl)octadecyl]-1,3-propanediol-3-phosphocholine(Compound XIV) utilized in Example 7. The imaging studies were performedon Dunning R3327 prostate tumor-bearing animals injected with >40 μCi ofthe radioiodinated phospholipid ether formulations described above. Theanesthetized animals were imaged at selected times after injection on agamma camera set to the ¹²⁵I window and outfitted with a low energycollimator. Static fifteen-minute images were obtained for each animalat the specified time. The results are illustrated in FIGS. 8A-8C.

As illustrated in FIGS. 8A-8C for 18-(p-iodophenyl)octadecylphosphocholine (Compound XVI), 12-(p-iodophenyl)dodecyl phosphocholineand 1-O-[18-(p-iodophenyl)octadecyl]-1,3-propanediol-3-phosphocholine(Compound XIV), respectively, the mass designated as T corresponds tothe tumor tissue. Administration of the C-18 compounds resulted inexcellent tumor localization in comparison with the C-12 analog, whereinthe tumor was barely visible.

In addition to the foregoing specifically mentioned uses of theinventive compounds, the compounds of the present invention may findapplicability as carrier molecules for radiosensitizers.Radiosensitizers are agents administered to sensitize tumor tissue tothe effects of externally applied radiation. Well knownradiosensitizers, such as misonidazole and metronidazole are substitutednitroimidazoles. Substitution of an electron-capturing moiety, such asnitroimidazole, for the iodophenyl moiety in the phospholipid etheranalogues of the present invention would permit tumor-localizedsensitization for radiation therapy.

In yet another proposed use, the phospholipid analogues of the presentinvention could incorporate boron containing substituents for use asboron-neutron activation therapeutic agents. These therapeutic agentsare administered using the stable isotope of the electron-capturingboron. External radiation activates the boron to create tissuedestructive activity.

Although the invention has been described in terms of specificembodiments and applications, persons skilled in this art can, in lightof this teaching, generate additional embodiments without exceeding thescope or departing from the spirit of the claimed invention. Inparticular, the methods of synthesis are merely illustrative and can bemodified by those of skill in the art for the production of varioussubstituted phospholipid ether analogues in accordance with theinvention. Moreover, other techniques of radio-tagging the analogues maybe employed. Of course, the invention contemplates any one of theortho-, meta- and para-isomers of iodobenzyl as the iodine-bearingmoiety.

In addition, while certain preferred embodiments of the improvedphosphilipid ether analogs described herein incorporate an 18-carbonalkyl chain into the compound, both longer and shorter chains are alsocontemplated to be within the scope of the present invention. In theirassociation with the plasma membrane of cells, the long aliphatic chainof the phospholipid ether analogs of the present invention presumablybecome buried in the hydrophobic phospholipid bilayer. Both NMR andx-ray diffraction studies have shown that the thickness of thishydrophobic phase of the lipid bilayer is between 35 and 40 Angstromsdepending on the type of phospholipid making up the membrane. SeeYeagle, P., Ed., “The Structure of Biological Membranes,” page 345, CRCPress, Boca Raton, Fla. (1991). Accordingly, based on the expectationthat hydrophobic chains as long as 40 Angstroms can be expected tointercalate into such lipid bilayers, the present invention contemplatesthat the extended alkyl chain can be up to 30 carbons in length.

Accordingly, it is to be understood that the drawings and descriptionsin this disclosure are proffered to facilitate the comprehension of theinvention and should not be construed to limit the scope thereof. Otherimprovements and modifications which become apparent to persons ofordinary skill in the art only after reading this disclosure, thedrawings and the following claims are deemed within the spirit and scopeof the present invention.

We claim:
 1. A compound of the general formula:

where X is a radioactive isotope of iodine; n is an integer between 16and 30; Y is selected from the group consisting of H, OH, COOH,

and OR, and Z is selected from the group consisting of NH₂, NR₂, andNR₃, wherein R is an alkyl or aralkyl substituent.
 2. The compound ofclaim 1 wherein X is a radioactive isotope of iodine consisting of ¹²³I,¹²⁵I, or ¹³¹I.
 3. A method of radio imaging a host, comprising the stepsof administering to the body of said host an effective amount of aradiopharmaceutical compound as claimed in claim 1 and subsequentlyscanning said host. 4.1-O-[18-(p-Iodophenyl)octadecyl]-1,3-propanediol-3-phosphocholine,wherein iodine is in the form of a radioactive isotope.
 5. The compoundof claim 4 wherein the radioactive isotope of iodine is selected fromthe group consisting of ¹²³I, ¹²⁵I, and ¹³¹I. 6.1-O-[18-(p-Iodophenyl)octadecyl]-2-O-methyl-rac-glycero-3-phosphocholine,wherein iodine is in the form of a radioactive isotope.
 7. The compoundof claim 6 wherein the radioactive isotope of iodine is selected fromthe group consisting of ¹²³I, ¹²⁵I, and ¹³¹I.